Eradication of smallpox as a natural pathogen happened 20 years ago. Eradication of smallpox as a biothreat is now our objective. New stockpiles of safe and efficacious smallpox vaccine are needed to protect both the civilian and military populations against deliberate release of the smallpox virus. Currently there is no commercially available vaccine, and the previously approved one is a live vaccinia inoculum associated with more adverse events than any other approved vaccine. Recent work has focused on improving the manufacturing process of the original vaccinia vaccine strain and testing other live attenuated viruses. We propose to discover new vaccine candidates from viral components. A subunit design would be safer and more easily controlled during manufacture. Since we anticipate obtaining multiple protective subunits, these can be mixed and matched to effectively defend against wild type, natural variants, and bioengineered smallpox isolates. To identify antigens of smallpox that carry vaccine potential, the goal of our proposed project is to screen all the genes of the closely related cowpox virus for their ability to protect against disease in its natural murine host. This genome-level approach is feasible because the viral genome databases are available and we have developed the platform technologies that make a comprehensive screen fast and efficient. We will establish the electronic and molecular protocols to synthetically generate a thousand codon-optimized gene sequences. It will be used to produce a library of high quality cowpox subgenes. An advanced library screening method employing multiplex arrays will enable us to screen all the subgenes for protection in one experiment. Vaccine candidates will be confirmed and immune characterized. Both the cowpox candidates and their variola homologs will be formatted three ways and evaluated in immune and cowpox protection assays. This project will uncover new subunit vaccine candidates against variola and prepare them for final validation in a primate challenge experiment.